The present invention relates generally to the manipulation of genetic materials and, more particularly, to the manufacture of specific DNA sequences useful in recombinant procedures to secure the production of urogastrone and polypeptide analogs thereof.
Incorporated by reference herein for the purpose of providing information pertinent to the prior art with respect to recombinant DNA techniques is co-owned, co-pending U.S. patent application Ser. No. 375,493, filed May 6, 1982, now U.S. Pat. No. 4,652,639, by Yitzhak Stabinsky, entitled "Manufacture and Expression of Structural Genes".
A component in human urine which inhibits gastric acid secretion was first described by Gray in 1939 [Gray, et al., Science, 89, 489 (1939)]. This component, named "urogastrone" was completely sequenced and its structure was published in 1975 [N. Gregory, Nature (London), 257, 325 (1975)]. Earlier, the isolation and characterization of a factor from mouse salivary glands which promotes the growth of epidermal tissue had been published [Cohen, J. Biol. Chem., 237, 1555 (1962)]. This compound was called "epidermal growth factor". When the amino acid composition of epidermal growth factor was compared with that of urogastrone, it was found that the two peptides were closely related. It is now known that these compounds, mouse and human epidermal growth factor-urogastrone (EGF-URO), are examples of a large class of "growth factors" and are widespread in animals and man.
EGF-URO like the other growth factors such as insulin, nerve growth factor, the insulin-like growth factors, and the like, is synthesized in mammals as part of a larger "pro-peptide" molecule from which it is cleaved by specific proteases to liberate the active form of the protein [Frey, et al., Proc. Nat. Acad. Sci., 76, 6294 (1979)]. When cleaved from its pro-peptide, EGF-URO, in both the mouse and in man, is composed of 53 amino acids. Further processing in the body also gives rise to a 51 amino acid-containing form which lacks the two amino acid residues at the carboxyl terminus of the peptide. The 53 and 51 amino acid forms of the peptide are called beta- and gamma- EGF-URO, respectively. Both forms have shown high activity as inhibitors of gastric acid secretion and as stimulators of growth of epidermoid tissue. High gastric secretion inhibitory activities have also been reported for the 46 and 47 amino acid products of selective enzymatic degradation. [See, U.S. Pat. Nos. 4,032,633 and 4,035,485.]
Receptors for EGF-URO have been found in various tissues of the human, mouse, rat, chicken, rabbit, cow, monkey, dog, cat, mink and hamster [Adamson, et al., Mol. Cell. Biochem., 34, 129 (1981)]. Work done with mouse and human EGF-URO has shown that they have identical activities in both species, the best documented of which are the abilities to virtually stop gastric acid secretion and to cause proliferation of epidermal and other epithelial tissues. [See, e.g, Starkey, et al., Science, 189, pp. 800-803 (1974) and Carpenter, Birth Defects: Original Article Series, 16, pp. 61-72 (1980)].
Despite its significant biological activities, little has been done to explore the full clinical potential of urogastrone and synthetic analogs thereof. This is due in large part to lack of large quantities of the substance. EGF-URO is presently isolated in small quantities by purification from mouse salivary glands or by a complex purification from human urine [Hollenberg, Vitamins and Hormones, 37, 69 (1979); Gregory et al., U.S. Pat. No. 3,883,497].
The polypeptide substance is too large to be readily synthesized by the well-known Merrifield procedure. Recombinant DNA techniques for the manufacture, cloning and expression of a structural gene for urogastrone and genes for polypeptide analogs which differ therefrom in terms of the identity and/or location of one or more amino acids have not been brought to bear on this problem.